RESUMO
We combined electrophysiological and freeze-fracture methods to estimate the unitary turnover rate of the gamma-aminobutyric acid (GABA) transporter GAT1. Human GAT1 was expressed in Xenopus laevis oocytes, and individual cells were used to measure and correlate the macroscopic rate of GABA transport and the total number of transporters in the plasma membrane. The two-electrode voltage-clamp method was used to measure the transporter-mediated macroscopic current evoked by GABA (I(NaCl)(GABA)), macroscopic charge movements (Q (NaCl)) evoked by voltage pulses and whole-cell capacitance. The same cells were then examined by freeze-fracture and electron microscopy in order to estimate the total number of GAT1 copies in the plasma membrane. GAT1 expression in the plasma membrane led to the appearance of a distinct population of 9-nm freeze-fracture particles which represented GAT1 dimers. There was a direct correlation between Q (NaCl) and the total number of transporters in the plasma membrane. This relationship yielded an apparent valence of 8 +/- 1 elementary charges per GAT1 particle. Assuming that the monomer is the functional unit, we obtained 4 +/- 1 elementary charges per GAT1 monomer. This information and the relationship between I(NaCl)(GABA) and Q (NaCl) were used to estimate a GAT1 unitary turnover rate of 15 +/- 2 s(-1) (21 degrees C, -50 mV). The temperature and voltage dependence of GAT1 were used to estimate the physiological turnover rate to be 79-93 s(-1) (37 degrees C, -50 to -90 mV).
